Synthesis of deuterium labeled NMDA receptor inhibitor - 20-Oxo-5β-[9,12,12-2H3]pregnan-3α-yl-l- glutamyl 1-ester

Article abstract of DOI:10.1016/j.steroids.2011.12.019

20-Oxo-5β-[9,12,12-²H₃]pregnan-3α-yl-l- glutamyl 1-ester 11 was synthesized as an internal standard for quantification of a neuroprotective NMDA receptor ligand, 20-oxo-5β-pregnan-3α-yl-l- glutamyl 1-ester 18 and its metabolites, in

Full text of DOI:10.1016/j.steroids.2011.12.019

Steroids 77 (2012) 282–287
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Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis of deuterium labeled NMDA receptor inhibitor –
20-Oxo-5b-[9,12,12-2H3]pregnan-3
a-yl-L-glutamyl 1-ester
c
c
Vojtech Kapras a,b, Alena Slavickova a, Eva Stastna a, , Ladislav Vyklicky Jr. , Karel Vales ,
⇑
Hana Chodounska a,d
a Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Flemingovo nam. 2, Prague 6 – Dejvice, Czech Republic
b Department of Organic and Nuclear Chemistry, Faculty of Science, Charles University, Prague 2, Czech Republic
c Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, Prague 4, Czech Republic
d Centre of the Region Hana for Biotechnological and Agricultural Research, Palacky University, Slechtitelu 11, Olomouc, Czech Republic
a r t i c l e i n f o
a b s t r a c t
20-Oxo-5b-[9,12,12-2H3]pregnan-3
for quantification of a neuroprotective NMDA receptor ligand, 20-oxo-5b-pregnan-3
1-ester 18 and its metabolites, in plasma and tissue. 11
a
-yl-
L
-glutamyl 1-ester 11 was synthesized as an internal standard
-yl- -glutamyl
-Hydroxy-progesterone (1) was reduced under
basic conditions to yield the corresponding 5b-steroid. Protection of the 3- and 20-oxo groups and
oxidation of the 11 -hydroxy group was then followed by a deuterium exchange, conducted under basic
conditions using deuterated methanol. Next, the carbonyl moiety at C-11 was reduced and the
11 -hydroxyl group removed through utilization of the Barton–McCombie reaction. Subsequent
Article history:
Received 31 October 2011
Received in revised form 9 December 2011
Accepted 12 December 2011
Available online 19 December 2011
a
L
a
a
Keywords:
a
NMDA receptor
Neuroprotection
Deuterium labeling
deprotection of the 3- and 20-acetals and stereoselective reduction of the 3-oxo group gave the desired
trideuterated pregnanolone (8). This was coupled with protected glutamic acid, which was then depro-
tected to yield [9,12,12-2H3]-pregnanolone glutamate (11) with >99% isotopic purity.
Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction
[3–6], their clinical utility is rather limited due to the severity of
their side effects [7].
Over the last 20 years, there has been a growing interest in neu-
rosteroids. These steroids are synthesized either de novo in the
brain or through the metabolic transformation of circulating
precursors. Neurosteroids exert their effects through modulation
of the synaptic and/or extrasynaptic neurotransmitter–receptors,
and are utilized by the brain for fine-tuning the function of the
neural network [1]. Often, excitatory transmissions are mediated
Of interest however, is the fact that progesterone and its
reduced metabolites also behave as endogenous neuroprotectives
[8,9]. In particular, 3a,5b-pregnanolone sulfate has been shown
to be an effective, use-dependent antagonist of the NMDA receptor
with a high neuroprotective potential [10]. Unfortunately, this
ligand is susceptible to the fast metabolic deactivation caused by
sulfatases [11]. Moreover, the bioavailability of this, and other
steroid sulfates is low. On the other hand, derivatives wherein
the sulfate moiety is substituted by glutamate or another charged
substituent have shown pharmacological promise, as they have
displayed neuroprotective effects in behavioral tests [12]. There-
fore, our focus is on the development of novel synthetic steroidal
inhibitors of the NMDA receptor that are resistant to sulfohydro-
by
L-glutamic acid, namely through the N-methyl-D-aspartate
receptor (NMDA) receptor. This receptor forms a Ca2+ permeable
ion channel, and under physiological conditions the activation of
this receptor is essential for long-term potentiation and basic cog-
nitive function, including learning and memory [2]. However, over-
excitation of the NMDA receptor can also induce cell death. This
excitotoxicity is often seen in conjunction with ischemia injuries
in the brain, and is also thought to contribute to the neurodegen-
eration associated with various forms of dementia [2]. While a
number of clinical studies have shown that NMDA receptor antag-
onists exhibit a neuroprotective effect in several human disorders
lase activity. As such, 3a,5b-pregnanolone glutamate 18 was
selected as our lead structure [12]. In order to examine its bioavail-
ability and pharmacokinetics, it was also necessary to synthesize
an isotopically labeled inner standard. This would allow for the
quantitative assessment of steroid 18 and its metabolites in tissues
and body fluids through the use of LC/MS. Theoretically, two stable
isotopes, 13C or deuterium (2H) could be used. The synthesis of
13C-labeled compounds is rather complex and expensive. In
contrast, the incorporation of a deuterium label is generally less
difficult and relatively inexpensive. Furthermore, an inner standard
⇑
Corresponding author. Address: Institute of Organic Chemistry and Biochem-
istry, Academy of Sciences of the Czech Republic, v.v.i., Flemingovo nam. 2, 166 10
Prague 6 – Dejvice, Czech Republic. Tel.: +420 220 183 316; fax: +420 220 183 578.
E-mail address: stastna@uochb.cas.cz (E. Stastna).
0039-128X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2011.12.019

V. Kapras et al. / Steroids 77 (2012) 282–287
283
molecule having deuterium labels differs by only 3–4 mass units
from the tested molecule [13], and therefore would not affect chro-
matographic separation nor would its MS peak overlap with the
63.30, 55.61, 50.59, 47.14, 45.84, 44.18, 42.70, 39.98, 38.43,
35.98, 34.42, 31.38, 26.83, 25.60, 24.36, 23.10, 23.03, 14.44.
C21H32O3 calcd: C, 75.86; H, 9.70. C21H32O3: calcd. C, 75.86; H,
natural isotopic steroid peaks. While a deuterated
L
-glutamic acid
9.70. Found. C, 75.76; H, 9.81. Compound 2b: mp 196–198 °C (lit.
is commercially available, its deuterium labels are adjacent to a
carbonyl group, and are consequently susceptible to deuterium/
proton exchange. Moreover, the isotopic labels are unsuitably
placed on a hydrolyzable substituent. However, deuterium labels
that are incorporated directly into the steroid core will remain in-
tact during chemical processes, analytical procedures, or metabolic
degradation.
198–201 °C [15]). 1H NMR d 0.66 s, 3 H (3 ꢁ H-18); 1.14 s, 3 H
(3 ꢁ H-19); 2.14 s, 3 H (3 ꢁ H-21); 2.55 t, 1 H, J1 = 9 (H-17
a);
2.74–2.79 m, 1 H (2b-H); 3.95–4.03 m, 1 H (H-11b). 13C NMR d
211.75, 208.79, 68.96, 63.29, 59.80, 55.59, 50.65, 47.35, 45.12,
44.07, 40.14, 38.31, 37.27, 34.51, 31.55, 31.36, 29.33, 24.39,
22.95, 14.41, 11.80. C21H32O3: calcd. C, 75.86; H, 9.70. Found. C,
75.77; H, 9.69.
The following paper describes an efficient synthesis of a trideu-
terated [9,12,12-2H3]-3
a
,5b-pregnanolone glutamate (3
d3, 11), containing deuterium atoms in positions 9 , 12
12b from the commercially available 11 -hydroxyprogesterone
a
5bP-Glu-
2.2.2. 3,20-Bis(ethylenedioxy)-5b-pregnan-11a-ol (3)
a
a, and
A mixture of dione 2a (4 g, 12.0 mmol), ethylene glycol (4.8 mL,
86.1 mmol), triethyl orthoformate (10.4 mL, 62.5 mmol), and p-tol-
uenesulfonic acid monohydrate (42 mg, 0.21 mmol) in absolute
benzene (90 mL) was stirred at room temperature overnight. Then,
the reaction mixture was poured into saturated solution of sodium
bicarbonate (70 mL) and the product was extracted with ethyl ace-
tate (3 ꢁ 50 mL). Combined organic extracts were washed with
solution of sodium bicarbonate (2 ꢁ 150 mL), water (2 ꢁ 150 mL),
and dried over sodium sulfate. Solvents were removed under vacuo
to afford product 3 (3.8 g, 75%) which was used further without
a
(1). Importantly, all three deuterium labels are located in specific
positions such that both the chemical and metabolic stability is
guaranteed.
2. Experimental
2.1. General methods
purification: [a]
+15 (c 0.28, CHCl3) (lit. +21 (c 1.06) [16]). 1H
D
Melting points were determined on a micro-melting point
apparatus Hund/Wetzlar (Germany) and are uncorrected. Optical
rotations were measured in chloroform using an Autopol IV
NMR d 0.76 s, 3 H (3 ꢁ H-18); 1.07 s, 3 H (3 ꢁ H-19); 1.29 s, 3 H
(3 ꢁ H-21); 3.86–4.01 m, 4 H (OCH2CH2O); 3.93 s, 4 H (OCH2-
CH2O); 4.11–4.15 m, 1 H (H-11b). C25H40O5: calcd. C, 71.39; H,
9.59. Found. C, 71.52; H, 9.42.
(Rudolf Research Analytical, Flanders, USA), [a] values are given
D
in ° (10ꢀ1 deg cm2 gꢀ1). IR spectra were recorded on a Bruker IFS
55 spectrometer (wavenumbers in cmꢀ1). Proton and carbon
NMR spectra were measured on a FT NMR spectrometer Bruker
AVANCE-400 (400, 100 MHz) in CDCl3 with tetramethylsilane as
the internal standard. Chemical shifts are given in ppm (delta
scale), coupling constants (J) and width of multiplets (W) are given
in Hz. Mass spectra were obtained with spectrometers ZAB-EQ
(at 70 eV) or LCQ Classic (Thermo Finnigan). Thin layer chromatog-
raphy (TLC) was performed on silica gel (ICN Biochemicals),
preparative TLC (prep-TLC) was carried out on 200 ꢁ 200 mm
plates coated with a 0.4 mm thick layer of the same material.
2.2.3. 3,20-Bis(ethylenedioxy)-5b-pregnan-11-one (4)
Pyridinium chlorochromate on aluminum oxide (2.5 g) was
added to a solution of compound 3 (2.5 g, 5.9 mmol) in benzene
(30 mL). Then, the reaction mixture was stirred at room tempera-
ture. After 24 h, pyridinium chlorochromate on aluminum oxide
(3 g) was added [17]. After 12 h, the solids were filtered off and
washed with benzene. Combined organic filtrates were evaporated
and the residue was crystallized from acetone/n-heptane to afford
white crystals of compound 4 (2.1 g, 84%): mp 124.5–126.5 °C (lit.
125.4–127 °C [18]). 1H NMR d 0.70 s, 3 H (3 ꢁ H-18); 1.17 s, 3 H
(3 ꢁ H-19); 1.25 s, 3 H (3 ꢁ H-21); 3.84-3.99 m, 4 H (OCH2CH2O),
3.92 s, 4 H (OCH2CH2O). C25H38O5: calcd. C, 71.74; H, 9.15. Found.
C, 71.57; H, 9.13.
For column chromatography, neutral silica gel 60 lm (Merck)
was used. Analytical samples were dried over phosphorus
pentoxide at 50 °C/100 Pa. Anhydrous THF was prepared by
distillation with benzophenone/Na immediately prior to use. An
aqueous solution of potassium bicarbonate was used as a satu-
rated solution.
2.2.4. 3,20-Bis(ethylenedioxy)-11b-hydroxy-5b-
[9,12,12-2H3]pregnane (5)
2.2. Synthesis of [9,12,12-2H3]-pregnanolone glutamate (11)
Potassium tert-butoxide (1.3 g, 11.6 mmol) was added to a solu-
tion of compound 4 (1 g, 2.4 mmol) in MeOD (490 mmol, 20 mL)
and tetrahydrofuran (30 mL). The reaction mixture was refluxed
under inert atmosphere for 13 days. Then, solvents were evapo-
rated under vacuum and deuterium oxide (2.5 mL) was added.
The supernatant was removed by pipette and the precipitate was
washed by another portion of deuterium oxide (2.5 mL). The resi-
due was dried by azeotropic evaporation with toluene
(3 ꢁ 10 mL) and then dissolved in freshly dry tetrahydrofuran
(10 mL). This solution of deuterated steroid was added dropwise
to the refluxing mixture of lithium aluminum hydride (1 g,
26.3 mmol) in tetrahydrofuran (20 mL) under inert atmosphere.
After 2 h, the reaction mixture was allowed to attain room temper-
ature and saturated aqueous solution of sodium sulfate (4 mL) was
added. The mixture was dried over the large excess of anhydrous
sodium sulfate. Precipitated aluminum salts were decanted and
washed with ether (3 ꢁ 10 mL). The combined solvents were
removed under reduced pressure and the residue was crystallized
from petroleum ether/ether to give white crystals of deuterated
2.2.1. 11a-Hydroxy-5b-pregnane-3,20-dione (2)
Palladium on CaCO3 (1.5 g, 5%) was added into a solution of
compound 1 (15 g, 45.1 mmol) in absolute pyridine (66 mL). The
reaction mixture was hydrogenated under slight overpressure of
hydrogen. After 8 h, the catalyst was filtered off and washed with
chloroform. Combined organic eluents concentrated in vacuo and
consecutively evaporated three times with toluene (3 ꢁ 20 mL).
The residue was then poured into diluted hydrochloric acid
(100 mL, 5%) and extracted with chloroform (2 ꢁ 60 mL). Com-
bined organic extracts were washed with water (50 mL), solution
of sodium bicarbonate (50 mL), and dried over sodium sulfate. Sol-
vents were removed under vacuum and the residue was purified
by column chromatography on silica gel (450 g, 50% ethyl acetate
in petroleum ether). Crystallization from acetone/n-heptane affor-
ded 6.9 g (46%) of compound 2a and 2.6 g (17%) of compound 2b.
Compound 2a: mp 124–126 °C (lit. 116–118 °C [14]). 1H NMR d
0.66 s, 3 H (3 ꢁ H-18); 1.14 s, 3 H (3 ꢁ H-19); 2.14 s, 3 H
(3 ꢁ H-21); 2.56 t, 1 H, J = 9 (H-17
a
); 2.67 t, 1 H, J = 14.5 (H-4
a
);
steroid
5
(950 mg, 94% yield, 99.4% isotopic purity): mp
3.96–4.04 m, 1 H (H-11b). 13C NMR d 213.78, 208.82, 68.69,
130–133 °C, [
a
]
D
+42.5 (c 0.15, CHCl3). 1H NMR d 0.97 s, 3 H

284
V. Kapras et al. / Steroids 77 (2012) 282–287
(3 ꢁ H-18); 1.20 s, 3 H (3 ꢁ H-19); 1.30 s, 3 H (3 ꢁ H-21); 3.74–
stirred ice-cold solution of compound 7 (506 mg, 1.6 mmol) in
methanol (37 mL). Then, an ice-cold solution of sodium borohy-
dride (80 mg, 2.1 mmol) in pyridine (11 mL) was added. The pro-
gress of the reaction was continuously checked by TLC. Once the
reaction was complete (15 min), it was quenched with 5% HCl
(pH checked). Resulting mixture was extracted with diethyl ether
(3 ꢁ 25 mL), and combined organic extracts were consecutively
washed with solution of sodium bicarbonate (25 mL) and brine.
Organic phase was dried with sodium sulfate and solvent
evaporated in vacuo. Chromatography on silica gel (20% ether in
4.00 m, 4 H (OCH2CH2O); 3.94 s, 4 H (OCH2CH2O); 4.14 s, 1 H (H-
11a
). 13C NMR d 111.90, 109.92, 68.48, 64.98, 64.25, 64.09, 63.26,
58.66, 57.85, 44.61, 43.24, 41.25, 35.77, 34.14, 30.83, 30.63,
26.76, 26.43, 25.95, 24.47, 23.76, 22.82, 15.73. IR (CHCl3) 1048
(C-OH), 1181 (CH2, OCH2CH2O), 1048, 1078, 1096 (ring, OCH2-
CH2O), 2101, 2198 (CD2), 1365, 1374, 1383 (CH3). MS (ESI): 446.3
(99.4%, M+Na); 445.3 (0.2% M(D2)+Na); 444.3 (0.3% M(D1)+Na);
443.3 (0.1% M(D0)+Na). C25H37D3O5: calcd. C, 70.88; H, 8.80. Found.
C, 70.98; H, 9.12.
petroleum ether) gave desired 3
128–130 °C, [
+86.0 (c 0.1, CHCl3). 1H NMR d 0.59 s, 3 H
(3 ꢁ H-18); 0.93 s, 3 H (3 ꢁ H-19); 2.11 s, 3 H (3 ꢁ H-21); 2.56 t,
1 H, J = 9.1 (H-17
); 3.61–3.68 m, 1 H (H-3). 13C NMR d 209.60,
a-alcohol 8 (253 mg, 50%): mp
2.2.5. 3,20-Bis(ethylenedioxy)-5b-[9,12,12-2H3]pregnan-11b-yl-O-(S-
methyldithiocarbonate) (6)
a]
D
A
solution of n-butyllithium (1.6 M in hexane, 1.5 mL,
a
2.4 mmol) was added dropwise to a stirred solution of alcohol 5
(832 mg, 2.0 mmol) in dry tetrahydrofuran (10 mL) at 0 °C (ice-
bath). The reaction mixture was allowed to attain room tempera-
ture. After 1 h, carbon disulfide (>99.9%, 0.365 mL, 6.04 mmol)
was added and the reaction mixture was stirred overnight at room
temperature. Then, methyl iodide (0.25 mL, 4.02 mmol) was added.
After 2 h, the reaction mixture was quenched with saturated so-
dium bicarbonate (50 mL). The product was extracted with diethyl
ether (3 ꢁ 15 mL), combined organic extracts were washed with
brine, dried over sodium sulfate, and the solvent was removed in
vacuo. Chromatography on silica gel (30 g) in petroleum ether-
ethyl acetate-triethylamine (80:20:1) gave xanthate 6 as a yellow
71.74, 63.86, 56.71, 44.16, 42.01, 36.42, 35.76, 35.35, 34.54,
31.48, 30.53, 27.07, 26.39, 24.44, 23.26, 22.89, 20.53, 13.34. IR
(CHCl3) 1036 (CAOH), 1698 (C@O), 2101, 2191 (CD2), 1384, 1378
(CH3). MS (ESI): 344.3 (98%, M+Na); 343.3 (1.9% M(D2)+Na);
342.3 (0.3% M(D1)+Na); 341.3 (0.1% M(D0)+Na). C21H31D3O2: calcd.
C, 78.45; H, 9.72. Found. C, 78.78; H, 10.10.
2.2.8. 20-Oxo-5b-[9,12,12-2H3]pregnan-3
[(tert-butoxycarbonyl)amino]-5-oxo-pentanoate (9)
Compound (245 mg, 0.8 mmol) and Boc-Glu(OBzl)-OH
a-yl (2S)-5-(benzyloxy)-2-
8
(257 mg, 0.8 mmol) were dissolved in dry benzene (8.5 mL). Then,
4-dimethylaminopyridine (10 mg, 0.1 mmol) and dicyclohexylcar-
bodiimide (1 M in benzene, 0.84 mL, 0.84 mmol) were added under
inert atmosphere and the reaction mixture was stirred at room
temperature. After 12 h, the reaction mixture was poured into sat-
urated solution of sodium bicarbonate (50 mL) and the product
was extracted with ethyl acetate (3 ꢁ 15 mL). Combined organic
extracts were washed with water (2 ꢁ 15 mL). Precipitated N,N0-
dicyclohexylurea was filtered off, filtrate was dried over anhydrous
sodium sulfate, and the solvent was removed in vacuo. Chromatog-
raphy on silica gel (15 g, 10% ether in petroleum ether) gave oily
foam (854 mg, 85%): [a]
+15.0 (c 0.11, CHCl3). 1H NMR d 0.70 s,
D
3 H (3 ꢁ H-18); 0.94 s, 3 H (3 ꢁ H-19); 1.13 s, 3 H (3 ꢁ H-21);
2.58 s, 3 H (3 ꢁ H-MeS); 3.73-3.90 m, 4 H (OCH2CH2O); 3.93 s, 4
H (OCH2CH2O); 6.03 s, 1 H (H-11a
). 13C NMR d 214.23, 111.62,
109.68, 80.56, 65.05, 64.25, 64.09, 63.32, 58.59, 57.57, 44.69,
43.17, 40.98, 35.73, 34.35, 31.58, 30.32, 26.46, 26.0, 25.80, 24.40,
23.66, 22.75, 18.88, 15.24. IR (CHCl3) 1245 (CAOAC, xanthate),
1054 (C@S), 1180 (CH2, OCH2CH2O), 2100, 2191 (CD2), 1373
(CH3). MS (ESI): 536.3 (99.4%, M+Na); 535.3 (0.2% M(D2)+Na);
534.3 (0.3% M(D1)+Na); 533.3 (0.1% M(D0)+Na). C27H39D3O5S2:
calcd. C, 63.12; H, 7.65; S, 12.48. Found. C, 62.98, H, 7.88; S, 12.72.
conjugate 9 (456 mg, 93%): [a]
+103 (c 0.12, CHCl3). 1H NMR d
D
0.60 s, 3 H (3 ꢁ H-18); 0.93 s, 3 H (3 ꢁ H-19); 1.42–1.45 m, 9 H
(3 ꢁ CH3-Boc); 2.12 s, 3 H (3 ꢁ H-21); 2.43–2.52 m, 2 H (CH2-40);
2.2.6. 5b-[9,12,12-2H3]Pregnane-3,20-dione (7)
2.54 t, 1 H, J = 8.8 (H-17a); 4.29 dd, 1 H, J1 = 8.1, J2 = 12.8 (Ha,
Tributyltin hydride (0.67 mL, 2.49 mmol) was added to a reflux-
ing solution of compound
CH-20); 4.73–4.80 m, 1 H (H-3b); 5.10–5.12 m, 1 H (NH); 5.13–
5.16 m, 2 H (CH2-benzyl); 7.34–7.38 m, 5 H (phenyl). 13C NMR d
209.40, 170.50, 170.15, 155.36, 135.22, 128.57 (2 ꢁ C), 128.26
(2 ꢁ C), 128.17, 80.13, 75.69, 66.46, 63.84, 56.61, 50.76, 44.14,
41.84, 35.71, 34.95, 34.54, 32.12, 31.47, 30.32, 28.32 (3 ꢁ C),
28.02, 26.86, 26.57, 26.26, 24.41, 23.18, 22.93, 20.56, 13.34. IR
(CHCl3) 1704 (C@O, acetate), 1357 (CAH, acetate), 2100, 2192
(CD2), 1731 (C@O, glutamate), 1714 (C@O, -NHBoc), 1499 (amide,
-NHBoc), 1232 (CAO, glutamate); 1384 (CH3). MS (ESI): 663.4
(98%, M+Na); 662.4 (1.9% M(D2)+Na); 661.4 (0.3% M(D1)+Na);
660.4 (0.1% M(D0)+Na). C38H52D3NO7: calcd. C, 71.22; H, 8.19; N,
2.19. Found. C, 71.31; H, 8.23; N, 2.16.
6
(848 mg, 1.7 mmol) and 1,10-
azobis(cyclohexanecarbonitrile) (81 mg, 0.33 mmol) in dry toluene
(50 mL) under argon atmosphere. After 4 h of reflux, tetrahydrofu-
ran was removed in vacuo and the oily residue was dissolved in
acetone (50 mL). Water (5 mL) and HCl (36%, 2 mL) were added
and the reaction mixture was stirred for 15 min. Then, the solvents
were partly evaporated (1/2) and the residue was poured into the
solution of sodium bicarbonate (100 mL). The product was
extracted with ethyl acetate (3 ꢁ 20 mL), washed with brine
(2 ꢁ 30 mL), and dried over magnesium sulfate. Solvent was
removed in vacuo and the residue was purified by a column chro-
matography on a silica gel (40 g, 10% ether in petroleum ether) to
afford 646 mg (quant.) of compound 7 which was contaminated
with traces of inseparable tributyltin residues. Compound 7 was
used without further purification. 1H NMR d 0.64 s, 3 H (3 ꢁ H-
18); 1.03 s, 3 H (3 ꢁ H-19); 2.13 s, 3 H (3 ꢁ H-21); 2.55 t, 1 H,
2.2.9. 20-Oxo-5b-[9,12,12-2H3]pregnan-3
carbonyl)- -glutamyl 1-ester (10)
a-yl N-(tert-butoxy-
L
Palladium on CaCO3 (45 mg, 5%) was added to the solution of
compound 9 (449 mg, 0.7 mmol) in absolute MeOH (8 mL). The
reaction mixture was hydrogenated under slight overpressure of
hydrogen. After 8 h, the reaction mixture was filtered to remove
the catalyst, which was washed with chloroform. Combined fil-
trates were evaporated in vacuo. The residue was dissolved in
ether. Evaporation in vacuo gave white foam of compound 10
J = 9 (H-17a
). 13C NMR d 212.95, 209.33, 63.75, 56.61, 44.17,
44.11, 42.30, 37.17, 36.96, 35.45, 34.85, 31.48, 26.52, 25.76,
24.40, 22.58, 22.57, 20.91, 13.37. IR (CHCl3) 1703 (C@O), 1358
(CH3), 2100, 2191 (CD2), 1384, 1358 (CH3). MS (ESI): 342.3
(99.4%, M+Na); 341.3 (0.2% M(D2)+Na); 340.3 (0.3% M(D1)+Na);
339.3 (0.1% M(D0)+Na).
(379 mg, 98%): [a]
+140 (c 0.22, CHCl3). 1H NMR d 0.60 s, 3 H
D
(3 ꢁ H-18); 0.94 s,
3
H
(3 ꢁ H-19); 1.43–1.46 m,
9
H
2.2.7. 3
a
-Hydroxy-5b-[9,12,12-2H3]pregnan-20-one (8)
(3 ꢁ CH3-Boc); 2.12 s, 3 H (3 ꢁ H-21); 2.44–2.48 m, 2 H (CH2-40);
An ice-cold solution of sodium hydroxide (44 mg, 1.1 mmol) in
water (0.4 mL) and methanol (12 mL) was added to a vigorously
2.55 t, 1 H, J = 8.8 (H-17a); 4.30 dd, 1 H, J1 = 7.8, J2 = 12.4 (Ha,
CH-20); 4.76–4.82 m, 1 H (H-3b); 5.20 d, 1 H, J = 7.8 (NH). 13C

V. Kapras et al. / Steroids 77 (2012) 282–287
285
NMR d 209.58, 176.58, 171.52, 151.61, 80.23, 75.86, 63.84, 56.62,
52.90, 44.16, 41.86, 35.72, 34.95, 34.55, 32.13, 31.46, 30.03, 28.31
(3 ꢁ C), 28.19, 26.88, 26.57, 26.26, 24.42, 23.19, 22.94, 20.57,
13.35. IR (CHCl3) 1739 (C@O, COOH), 1701 (C@O, acetate), 1357
(CAH, acetate), 2101, 2192 (CD2), 1730 (C@O, glutamate), 1712
(C@O, NHBoc), 1501 (amide, NHBoc), 1378, 1384 (CH3). MS (ESI):
573.3 (98%, M+Na); 572.3 (1.9%, M(D2)+Na); 571.3 (0.3%,
M(D1)+Na); 570.3 (0.1%, M(D0)+Na). C31H46 D3NO7: calcd. C,
67.61; H, 8.44; N, 2.54. Found. C, 67.75; H, 8.39; N, 2.63.
56.31, 43.12, 43.08, 42.94, 42.89, 35.71, 34.32, 32.05, 31.41,
30.34, 26.53, 25.97, 25.71, 24.29, 23.43, 22.66, 19.02, 15.48. IR
(CHCl3) 1244 (CAOAC, xanthate), 1054 (C@S), 1179 (CH2, OCH2-
CH2O), 1373 (CH3). C27H42O5S2: calcd. C, 63.49; H, 8.29. Found. C,
63.86; H, 8.48.
2.3.3. 5b-Pregnane-3,20-dione (14)
Compound 14 was prepared in the same manner as compound
7 (Section 2.2.6). Starting from compound 13 (850 mg, 1.7 mmol),
compound 14 (808 mg, 95%) was obtained as a white solid: mp
118.6–119 °C (ethyl acetate/n-heptane) (lit. 118.5–120 °C [19]).
1H NMR d 0.64 s, 3 H (3 ꢁ H-18); 1.03 s, 3 H (3 ꢁ H-19); 2.13 s, 3
2.2.10. 20-Oxo-5b-[9,12,12-2H3]pregnan-3
(11)
a-yl L-glutamyl 1-ester
Trifluoroacetic acid (0.83 mL, 11.2 mmol) was added dropwise
to a stirred solution of compound 10 (363 mg, 0.7 mmol) in dichlo-
romethane (6 mL). After 2 h of stirring at room temperature and
then 12 h at 5 °C, solvents were removed by azeotropic distillation
with benzene. The residue was dissolved in pyridine (1 mL) and
MeOH (1 mL), solvents were evaporated. The crude product was
dissolved in chloroform (15 mL), washed with water, and dried
with anhydrous sodium sulfate. Solvent was removed in vacuo
and the oily residue was transformed into a white foam of preg-
nanolone glutamate 11 (245 mg, 82%) by dissolving in ether and
evaporating under reduced pressure at low temperature of the ro-
H (3 ꢁ H-21); 2.55 t, 1 H, J = 9 (H-17
a). C21H32O2: calcd. C, 79.70;
H, 10.19. Found. C, 79.73; H, 10.42.
2.3.4. 3a-Hydroxy-5b-pregnan-20-one (15)
Compound 15 was prepared in the same manner as compound
8 (Section 2.2.7). Starting from compound 14 (800 mg, 2.5 mmol),
compound 15 (408 mg, 50%) was obtained as a white solid after
column chromatography on silica gel (20% ether in petroleum
ether): mp 144–148 °C (lit. 146–148 °C [20]). 1H NMR d 0.60 s, 3
H (3 ꢁ H-18); 0.93 s, 3 H (3 ꢁ H-19); 2.11 s, 3 H (3 ꢁ H-21); 2.53
tary evaporator bath (15 °C): [
0.61 s, 3 H (3 ꢁ H-18); 0.96 s, 3 H (3 ꢁ H-19); 2.14 s, 3 H (3 ꢁ H-
21); 2.49 t, 2 H, J = 8.8 (CH2-40); 2.59 t, 1 H, J = 8.8 (H-17
); 3.73
a]
+108 (c 0.1, CHCl3). 1H NMR d
t, 1 H, J = 9.1 (H-17a); 3.60–3.68 m, 1 H (H-3). C21H34O2: calcd. C,
D
79.19; H, 10.76. Found. C, 79.10; H, 10.71.
a
dd, 1 H, J1 = 3.8, J2 = 7.8 (Ha, CH-20); 4.78–4.88 m, H (H-3b). 13C
NMR d 209.43, 177.53, 171.37, 75.82, 63.85, 56.66, 55.49, 44.29,
41.82, 40.46, 39.16, 35.71, 34.91, 34.61, 32.10, 31.47, 26.85,
26.53, 26.25, 24.40, 23.21, 22.93, 20.86, 13.40. IR (CHCl3) 1739
(C@O, COOH), 1700 (C@O, acetate), 1359 (CAH, acetate), 2099,
2192 (CD2), 1729 (C@O, glutamate), 3375 (NH2). MS (ESI): 473.4
(98%, M+Na); 472.4 (1.9% M(D2)+Na); 471.4 (0.3% M(D1)+Na);
470.4 (0.1% M(D0)+Na). C26H38 D3NO5: calcd. C, 69.30; H, 8.51; N,
3.11. Found. C, 69.31; H, 8.53; N, 3.02.
2.3.5. 20-Oxo-5b-pregnan-3a-yl (2S)-5-(benzyloxy)-2-[(tert-
butoxycarbonyl)amino]-5-oxo-pentanoate (16)
Compound 16 was prepared in the same manner as compound
9 (Section 2.2.8). Starting from compound 15 (250 mg, 0.8 mmol),
compound 16 (456 mg, 93%) was obtained as an oily material after
column chromatography on silica gel (10% ether in petroleum
ether):
[
a]
D
+38.0 (c 0.11, CHCl3). 1H NMR
d 0.60 s, 3 H
(3 ꢁ H-18); 0.93 s, 3 H (3 ꢁ H-19); 1.42–1.45 m, 9 H (3 ꢁ CH3-
Boc); 2.12 s, 3 H (3 ꢁ H-21); 2.43–2.47 m, 2 H (CH2-40); 2.53 t, 1
H, J = 8.8 (H-17a
); 4.3 dd, 1 H, J1 = 8.1, J2 = 12.8 (Ha, CH-20); 4.73–
2.3. Synthesis of pregnanolone glutamate (18)
4.78 m, 1 H (H-3b); 5.08–5.10 m, 1 H (NH); 5.13–5.18 m, 2 H
(CH2-benzyl); 7.35–7.40 m, 5 H (phenyl). 13C NMR d 209.60,
170.70, 170.35, 155.36, 135.42, 128.55 (2 ꢁ C), 128.38 (2 ꢁ C),
128.31, 80.02, 75.89, 66.71, 63.84, 56.62, 50.19, 44.29, 41.77,
40.38, 39.14, 36.94, 35.74, 34.89, 34.57, 31.98, 31.53 (2 ꢁ C),
28.30 (3 ꢁ C), 26.82, 26.32, 26.25, 24.38, 23.23, 22.87, 20.82,
13.40. IR (CHCl3) 1704 (C@O, acetate), 1357 (CH3, acetate), 3435
(NH, NHBoc), 1704 (C@O, NHBoc), 1499 (amide, NHBoc), 2979
(CH3, NHBoc), 1731 (C@O, glutamate), 1452 (ring, benzyl), 1330
(CH2, benzyl), 1386 (CH3). C38H55NO7: calcd. C, 71.55; H, 8.69; N,
2.20. Found. C, 71.62; H, 9.01; N, 2.37.
2.3.1. 3,20-Bis(ethylenedioxy)-5b-pregnan-11b-ol (12)
Lithium aluminum hydride (80 mg, 2.1 mmol) was added in
portions to a solution of compound 4 (100 mg, 0.2 mmol) in tetra-
hydrofuran (6 mL) and the reaction mixture was refluxed. After 2 h,
saturated solution of sodium sulfate (2 mL) was added. The mix-
ture was dried over large excess of anhydrous sodium sulfate.
The solvent was decanted from aluminum salts, washing with
ether (3 ꢁ 10 mL). The solvents were removed under reduced pres-
sure and crystallized from petroleum ether/diethyl ether to give
white plates of compound 12 (96 mg, 96%): mp 138–139.5 °C (lit.
138.5–139.5 °C [18]). 1H NMR d 0.97 s, 3 H (3 ꢁ H-18); 1.00–1.08
m, 1 H (H-9
1.38–1.50 m, 1 H (H-12
(H-12b); 3.85–3.99 m, 4 H (OCH2CH2O); 4.16–4.19 m, 1 H (H-
11 ). C25H40O5: calcd. C, 71.39; H, 9.59. Found. C, 71.17; H, 9.65.
a
); 1.2 s, 3 H (3 ꢁ H-19); 1.29 s, 3 H (3 ꢁ H-21);
2.3.6. 20-Oxo-5b-pregnan-3a-yl N-(tert-butoxycarbonyl)-L-glutamyl
a
); 2.18-2.23 dd, 1 H, J1 = 2.5, J2 = 14.1
1-ester (17)
Compound 17 was prepared in the same manner as compound
10 (Section 2.2.9). Starting from compound 16 (200 mg, 0.3 mmol),
a
compound 17 (171 mg, 99%) was obtained as white foam: [a]
D
2.3.2. 3,20-Bis(ethylenedioxy)-5b-pregnan-11b-yl-O-(S-methyl-
dithiocarbonate) (13)
+62.9 (c 0.08, CHCl3). 1H NMR d 0.60 s, 3 H (3 ꢁ H-18); 0.94 s, 3
H (3 ꢁ H-19); 2.12 s, 3 H (3 ꢁ H-21); 2.46 t, 2 H, J = 8.8 (CH2-40);
Compound 13 was prepared in the same manner as compound
6 (Section 2.2.5). Starting from compound 12 (250 mg, 0.6 mmol),
compound 13 (252 mg, 84%) was obtained as an oily material after
column chromatography on silica gel (petroleum ether/ethyl ace-
2.55 t, 1 H, J = 8.8 (H-17a); 4.3 dd, 1 H, J1 = 8.0, J2 = 12.4
(Ha, CH-20); 4.76–4.81 m, 1 H (H-3b); 5.20 d, 1 H, J = 7.8 (NH). 13C
NMR d 209.83, 175.22, 170.31, 155.45, 80.20, 75.67, 63.86, 56.63,
49.99, 44.33, 41.80 (2 ꢁ C), 39.13, 36.61, 35.76, 34.91, 34.60, 32.0,
31.51, 28.30 (3 ꢁ C), 26.86, 26.39, 26.26, 25.25, 24.40, 23.25,
22.88, 20.83, 13.40. IR (CHCl3) 1710 (C@O, acetate), 1357 (CH3,
acetate), 3434 (NH, NHBoc), 2979 (CH3, NHBoc), 1736 (C@O, gluta-
mate, COOH), 3511 (OH, COOH), 1736, 1710 (C@O, COOH), 1486
(CH3). C31H49NO7: cacld: C, 67.98; H, 9.02; N, 2.56. Found. C,
67.62, H, 8.72; N, 2.49.
tate/triethylamine, 80:20:1): [
0.74 s, 3 H (3 ꢁ H-18); 0.94 s, 3 H (3 ꢁ H-19); 1.13 s, 3 H
(3 ꢁ H-21); 1.51–1.60 m, 1 H (H-12 ); 1.73–1.79 m, 1 H (H-9 );
2.31–2.34 dd, 1 H, J1 = 2.5, J2 = 14.6 (H-12b); 2.55 s, 3 H (MeS);
3.72–3.88 m, 4 H (OCH2CH2O); 5.87 s, 1 H (H-11
). 13C NMR d
214.49, 111.53, 109.09, 79.81, 64.27, 64.13, 63.82, 63.71, 57.88,
a]
+11.1 (c 0.27, CHCl3). 1H NMR d
D
a
a
a

286
V. Kapras et al. / Steroids 77 (2012) 282–287
2.3.7. 20-Oxo-5b-pregnan-3
a
-yl
L
-glutamyl 1-ester (18)
of the deuterium labels to only the C-9 and C-12 positions. As
the acetal protecting group is acid-sensitive, the oxidation of the
11-hydroxyl group (3) was achieved by treatment with a mild oxi-
dative reagent – pyridinium chlorochromate on aluminum oxide.
Compounds 3 and 4 were prepared in yields of 75% and 84%,
respectively. The three deuterium labels were then introduced by
the refluxing of compound 4 with deuterated methanol and dry
THF in the presence of potassium tert-butoxide for 13 days. The
extensive reaction time was necessary due to the slower introduc-
tion of the deuterium label at the more sterically hindered 12b po-
sition, as preliminary experiments showed that deuterium
Compound 18 was prepared in the same manner as compound
11 (Section 2.2.10). Starting from compound 17 (200 mg,
0.4 mmol), compound 18 (159 mg, 98%) was obtained as white
foam: [
a
]
+106.7 (c 0.12, CHCl3). 1H NMR d 0.61 s, 3 H (3 ꢁ H-
D
18); 0.96 s, 3 H (3 ꢁ H-19); 2.13 s, 3 H (3 ꢁ H-21); 2.46 t, 2 H,
J = 8.8 (CH2-40); 2.59 t, 1 H, J = 8.8 (H-17
a); 3.61 dd, 1 H, J1 = 3.8,
J2 = 8.8 (Ha, CH-20); 4.78–4.86 m, 1 H (H-3b). 13C NMR d 210.95,
177.67, 170.59, 76.43, 63.59, 56.35, 53.08, 44.18, 41.56, 40.15,
38.78, 35.50, 34.54, 34.31, 33.25, 31.72, 31.14, 26.97, 26.56,
26.19, 25.96, 24.08, 22.85, 22.54, 20.56, 13.03. IR (CHCl3) 3516
(OH, COOH), 1739 (C@O, COOH), 1702 (C@O, acetate), 1359
(CAH, acetate), 1729 (C@O, glutamate), 3376 (NH2). C26H41NO5:
calcd. C, 69.77; H, 9.19; N, 3.11. Found. C, 69.56; H, 9.19; N, 2.99.
exchange in the less sterically hindered 9
a and 12a positions
was completed within hours. Next, the 11-oxo group was reduced
using lithium aluminum hydride to yield compound 5 in 99.4% iso-
topic purity (94% yield); with the isotopic distribution as follows:
99.4% 2H3 (m/z 446.3, M+Na), 0.2% 2H2 (m/z 445.3, M+Na), 0.3%
2H1 (m/z 444.3, M+Na), 2H0 (m/z 443.3, M+Na). Since position C-
11 is extremely sterically hindered, it was not surprising that initial
attempts to deoxygenate this position by refluxing an 11b-mesy-
late with LAH, or reducing an 11-tosylhydrazone with sodium
borohydride failed. Eventually, the deoxygenation was achieved
by the Barton–McCombie reaction [23,24] via xanthate intermedi-
ate 6, using ABCN for initiation and Bu3SnH as the hydrogen radical
source. Then, the deprotection of acetals was achieved by treat-
ment of compound 6 with hydrochloric acid in water-acetone mix-
ture. The 3-oxo group of compound 7 was selectively reduced
utilizing sodium borohydride at low temperature under slightly
basic conditions that suppress the reduction of sterically more hin-
dered 20-oxo group. The equatorial-3-alcohol 8 was formed as the
major product in 50% isolated yield. This corresponds to a known
preference of sodium borohydride for axial attack of the conform-
ationally-locked cyclohexanone systems. The equatorial configura-
tion was confirmed by 1H NMR spectroscopy (broad multiplet at
3.65 ppm). The last three steps of the synthesis pertain to the
introduction of the glutamyl ester moiety to position C-3. First,
compound 8 was esterified with Boc-Glu(OBzl)-OH using DCC
and DMAP. Secondly, the benzyl ether protecting group was
3. Results and discussion
The synthesis of [9,12,12-2H3]-3
(11) and 3 ,5b-pregnanolone glutamate (18) is summarized in
Scheme 1. The starting material for the synthesis was the commer-
cially available 11
-hydroxyprogesterone 1. The d4 double bond
was hydrogenated under basic conditions to give the 5b-derivative
2a (46% yield). In addition, the 5 -derivative 2b was also obtained
a,5b-pregnanolone glutamate
a
a
a
as the minor product (2.6 g, 17%). Their configurations at 5-H were
determined by comparison of melting points with literature
[14,15] as well as the 1H and 13C spectra. Simple discrimination be-
tween 5
plished by circular dichroism spectra [21]. The Cotton effect of
-alfa isomers is positive, while that of 5b-isomers is negative.
a- and 5b-isomers of 3-oxo-steroids used to be accom-
5a
However, we used more common NMR technique. There is a differ-
ence between the signals of hydrogen atoms in the position 4: the
1H NMR of the 5b-derivative 2a displays a characteristic pseudotri-
plet of the axial-4
a-H at d 2.67 ppm with splitting 14.5 Hz. On con-
trary, the 5 -derivative 2b is characterized by similar shape of the
a
axial-4b-H but shifted to higher fields (d ꢂ 2.4 ppm), where it is
overlapped by other signals [22]. Carbonyl moieties in positions
C-3 and C-20 were protected as acetals to limit the introduction
O
O
HO
O
HO
O
HO
O
O
H
O
H
H
H
H
H
H
H
a
b
c
H
H
H
H
H
H
H
H
O
O
O
O
H
O
H
O
H
2a: R = 5β-H
2b: R = 5α-H
4
1
3
S
R3
O
R3
R3
O
R3
R2
R1
R2
R1
R2
S
R2
R1
HO
O
O
O
d,e (for 5)
e (for 12)
O
O
H
H
g, h
H
H
H
H
f
i
H
H
R1
H
H
H
H
O
O
HO
O
O
H
H
O
H
H
5: R1=R2=R3= 2H
12: R1=R2=R3= H
8: R1=R2=R3= 2H
15: R1=R2=R3= H
6: R1=R2=R3= 2H
13: R1=R2=R3= H
7: R1=R2=R3= 2H
14: R1=R2=R3= H
R2 R3
H
R2 R3
H
R2 R3
O
O
O
H
H
H
j
H
k
l, m
O
O
O
R1
H
R1
H
R1
H
H2N
BocHN
BocHN
O
O
O
H
H
H
COOH
COOH
COOBzl
9: R1=R2=R3= 2H
16: R1=R2=R3= H
11: R1=R2=R3= 2H
18: R1=R2=R3= H
10: R1=R2=R3= 2H
17: R1=R2=R3= H
Scheme 1. (a) H2, Pd/CaCO3, pyridine; (b) ethylene glycol, triethyl orthoformate, p-TsOH, benzene; (c) PCC/Al2O3, benzene; (d) tert-BuOK, MeOD, THF, rfl; (e) LAH, THF, rfl; (f)
n-BuLi, CS2, CH3I, THF, rt; (g) ABCN, Bu3SnH, toluene, rfl; (h) HCl, water, acetone; (i) NaBH4, NaOH, MeOH, H2O, pyridine; (j) Boc-Glu(OBzl)-OH, DCC, DMAP, benzene; (k) H2,
Pd/CaCO3, MeOH; (l) CF3COOH, DCM; (m) pyridine, MeOH.

V. Kapras et al. / Steroids 77 (2012) 282–287
287
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were prepared in yields of 93%, 98%, and 82%, respectively.
Excluding the introduction of the three deuterium labels into the
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synthesis of compound 18. While this was an unusual and some-
what complex synthetic approach for the synthesis of compound
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the proposed synthetic strategy for compound 11 could be exam-
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followed by the Barton–McCombie deoxygenation, which afforded
xanthate 13 in 84% yield. Deprotection of acetal protecting groups
was achieved by treatment with hydrochloric acid in water-acetone
mixture. Compound 14 was prepared in 95% yield. Selective reduc-
tion of the 3-oxo group gave the equatorial-3-alcohol 15 (50% yield).
Finally, esterification with Boc-Glu(OBzl)-OH, followed by depro-
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successfully used in evaluating 3
and tissue [12]. This approach can also be expanded to the deute-
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three non-exchangeable deuterium labels (although, in this case
the introduction of a cis-A,B-ring fusion would increase the overall
number of synthetic steps) [25]. To the best of our knowledge, the
successful introduction of deuterium labels into the non-exchange-
a
5bP-Glu-d3 (11) was prepared and was
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able 9a, 12a, and 12b positions on the steroid skeleton has not
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Steroiden. Über Steroide. Helv Chim Acta 1961;44:186–98.
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a-oxido-
been described in the literature. In turn, this is a particularly
attractive strategy for the synthesis of novel deuterium labeled ste-
roids. In addition, this methodology could also provide useful start-
ing materials for investigating various future avenues in the field of
steroid chemistry.
Acknowledgments
ˇ
We are indebted to Dr. Vašícková for taking IR spectra and Dr.
P. Fiedler for interpreting them. Optical rotations were measured
and elemental analyses were carried out in the Analytical Labora-
[21] Šídová R, Stránsky´ K, Kasal A, Slavíková B, Kohout L. Long-range effect of 17-
substituents in 3-oxo steroids on 4,5-double bond hydrogenation. Coll Czech
Chem Commun 1998;63(10):1528–42.
ˇ
tory (Dr. S. Matejková, Head). The authors thank Dr. Laurel Lasser
for language corrections. This work was supported by funding from
the Grants IGA Ministry of Health NS10365, Research Projects of
the AS CR AV0Z 50110509, Z4 055 0506, and the Ministry of
Education, Youth and Sports of the Czech Republic (LC 06077,
1M0157, LC554, ED0007/01/01).
ˇ
5
´
[22] Chodounská H, Budešínsky M, Šídová R, Šíša M, Kasal A, Kohout L. Simple NMR
determination of /5b-configuration of 3-oxosteroids. Coll Czech Chem
a
Commun 2001;66(10):1529–44.
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procedure for the efficient deoxygenation of secondary alcohols. Regiospecific
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